László Virág

Peroxynitrite-induced thymocyte apoptosis: The role of caspases and poly (ADP-ribose) synthetase (PARS) activation

The mechanisms by which immature thymocyte apoptosis is induced during negative selection are poorly defined. Reports demonstrated that cross‐linking of T‐cell receptor leads to stromal cell activation, expression of inducible nitric oxide synthase (iNOS) and, subsequently, to thymocyte apoptosis. Therefore we examined, whether NO directly or indirectly, through peroxynitrite formation, causes thymocyte apoptosis. Immuno‐histochemical detection of nitrotyrosine revealed in vivo peroxynitrite formation in the thymi of naive mice. Nitrotyrosine, the footprint of peroxynitrite, was predominantly found in the corticomedullary junction and the medulla of naive mice. In the thymi of mice deficient in the inducible isoform of nitric oxide synthase, considerably less nitrotyrosine was found. Exposure of thymocytes in vitro to low concentrations (10u2003μm) of peroxynitrite led to apoptosis, whereas higher concentrations (50u2003μm) resulted in intense cell death with the characteristics of necrosis. We also investigated the effect of poly (ADP‐ribose) synthetase (PARS) inhibition on thymocyte apoptosis. Using the PARS inhibitor 3‐aminobenzamide (3‐AB), or thymocytes from PARS‐deficient animals, we established that PARS determines the fate of thymocyte death. Suppression of cellular ATP levels, and the cellular necrosis in response to peroxynitrite were prevented by PARS inhibition. Therefore, in the absence of PARS, cells are diverted towards the pathway of apoptotic cell death. Similar results were obtained with H2O2 treatment, while apoptosis induced by non‐oxidative stimuli such as dexamethasone or anti‐FAS antibody was unaffected by PARS inhibition. In conclusion, we propose that peroxynitrite‐induced apoptosis may play a role in the process of thymocyte negative selection. Furthermore, we propose that the physiological role of PARS cleavage by apopain during apoptosis may serve as an energy‐conserving step, enabling the cell to complete the process of apoptosis.

Suppression of macrophage inflammatory protein (MIP)-1α production and collagen-induced arthritis by adenosine receptor agonists

1 Ligands of the various adenosine receptor subtypes modulate the production of pro‐and anti‐inflammatory cytokines. Here we evaluated the effect of adenosine and various ligands of the adenosine receptor subtypes (A1, A2, A3) on the chemokine macrophage inflammatory protein (MIP) 1α production in immunostimulated RAW macrophages in vitro. Furthermore, we studied whether a selected A3 adenosine receptor agonist inhibits MIP‐1α production and affects the course of inflammation in collagen‐induced arthritis. 2 In the cultured macrophages, the A3 receptor agonist N6‐(3‐iodobenzyl)‐adenosine‐5′‐N‐methyluronamide (IB‐MECA), and, less potently, the A2 receptor agonist 2‐p‐(2‐carboxyethyl) phenethylamino‐5′‐N‐ethyl‐carboxamidoadenosine (CGS; 1–200u2003μM) dose‐dependently suppressed the production of MIP‐1α. The selective A1 receptor agonist 2‐chloro‐N6‐cyclopentyladenosine (CCPA, 1–200u2003μM) was ineffective, and adenosine was a weak inhibitor. The inhibition of MIP‐1α production by the A3 and A2 agonist was associated with suppression of its steady‐state mRNA levels. 3 Based on the in vitro data, we concluded that activation of A3, and to a lesser extent A2 adenosine receptors suppresses MIP‐1α expression. Since IB‐MECA was the most potent inhibitor of MIP‐1α expression, we next investigated whether it affects the production of other pro‐inflammatory mediators. We observed that IB‐MECA (1–300u2003μM) inhibited, in a dose‐dependent manner, the production of IL‐12, IL‐6, and, to a lesser extent, nitric oxide in the immunostimulated cultured macrophages. 4 Since MIP‐α is a chemokine which enhances neutrophil recruitment into inflammatory sites, we investigated whether the A3 agonist IB‐MECA affects the course of inflammation, MIP‐α production and the degree of neutrophil recruitment in arthritis. In a model of collagen‐induced arthritis in mice, IB‐MECA (0.5u2003mg/kg/day) reduced the severity of joint inflammation. IB‐MECA inhibited the formation of MIP‐1α, IL‐12 and nitrotyrosine (an indicator of reactive nitrogen species) in the paws, and suppressed neutrophil infiltration. 5 We conclude that adenosine receptor agonists, most notably the A3 agonist IB‐MECA suppress the production of MIP‐α, and exert anti‐inflammatory effects. Therefore, stimulation of adenosine receptor subtypes A3 and A2 may be a strategy worthy of further evaluation for the abrogation of acute or chronic inflammatory disorders.

Crucial role of apopain in the peroxynitrite-induced apoptotic DNA fragmentation

Peroxynitrite, a cytotoxic oxidant formed in the reaction of superoxide and nitric oxide is known to cause programmed cell death. However, the mechanisms of peroxynitrite-induced apoptosis are poorly defined. The present study was designed to characterize the molecular mechanisms by which peroxynitrite induces apoptosis in HL-60 cells, with special emphasis on the role of caspases. Peroxynitrite induced the activation of apopain/caspase-3, but not ICE/caspase-1 as measured by the cleavage of fluorogenic peptides. Considering the short half-life of peroxynitrite and the kinetics of caspase-3 activation (starting 3-4 h after peroxynitrite treatment), the enzyme is not likely to become activated directly by the oxidant. Caspase-3 activation proved to be essential for DNA fragmentation, because pretreatment of the cells with the specific tetrapeptide inhibitor DEVD-fmk completely blocked peroxynitrite-induced DNA fragmentation. Peroxynitrite-induced cytotoxicity was also significantly altered by the inhibition of caspase-3, whereas phosphatidylserine exposure was unaffected by DEVD-fmk treatment. Because many of the effects of peroxynitrite are mediated by poly(ADP-ribose) synthetase (PARS) activation, we have also investigated the effect of PARS-inhibition on peroxynitrite-induced apoptosis. We have found that PARS-inhibition modulates peroxynitrite-induced apoptotic DNA fragmentation in the HL-60 cells. The effect of the PARS inhibitors, 3-aminobenzamide and 5-iodo-6-amino-1,2-benzopyrone were dependent on the concentration of peroxynitrite used. While PARS-inhibition resulted in increased DNA-fragmentation at low doses (15 microM) of peroxynitrite, a decreased DNA-fragmentation was found at high doses (60 microM) of peroxynitrite. PARS inhibition negatively affected viability as determined by flow cytometry. These data demonstrate the crucial role of caspase-3 in mediating apoptotic DNA fragmentation in HL-60 cells exposed to peroxynitrite.

The crucial role of IL-10 in the suppression of the immunological response in mice exposed to staphylococcal enterotoxin B

Staphylococcal enterotoxin B (SEB), a bacterial superantigen, activates the immune system resulting in a burst of pro‐ and anti‐inflammatory cytokines. A central anti‐inflammatory mediator in this process is IL‐10. Using IL‐10‐deficient C57BL/6 (IL‐10 KO) mice, we studied the role of endogenous IL‐10 in the regulation of the immune response to SEB. SEB (100 μg) induced the release of IL‐10 in control C57BL/6 [IL‐10 wild type (WT)] mice, but not in their IL‐10 KO counterparts. SEB‐evoked plasma levels of TNF‐α, IL‐1β, IL‐2, IL‐6, IL‐12 and IFN‐γ were significantly higher in the IL‐10 KO mice than in the WT animals. The release of macrophage inflammatory proteins‐1α and −2 was also enhanced in the IL‐10 KO mice. Further, upon SEB challenge, mice deficient in IL‐10 produced higher levels of nitric oxide than the WT animals. IL‐10 deficiency resulted in a marked enhancement of the SEB‐induced apoptosis of thymocytes. Finally, IL‐10 KO mice were more susceptible to SEB‐induced lethal shock than their WT controls. These results show that IL‐10 plays an important immunoregulatory role in the response to a superantigenic stimulus, by dampening of the shock‐inducing inflammatory response and early activation‐induced cell death elicited by SEB.

Inhibition of poly(ADP-ribose) synthetase (PARS) and protection against peroxynitrite-induced cytotoxicity by zinc chelation

Peroxynitrite, a potent oxidant formed by the reaction of nitric oxide and superoxide causes thymocyte necrosis, in part, via activation of the nuclear enzyme poly(ADP‐ribose) synthetase (PARS). The cytotoxic PARS pathway initiated by DNA strand breaks and excessive PARS activation has been shown to deplete cellular energy pools, leading to cell necrosis. Here we have investigated the effect of tetrakis‐(2‐pyridylmethyl)‐ethylenediamine (TPEN) a heavy metal chelator on peroxynitrite‐induced cytotoxicity. TPEN (10u2003μM) abolished cell death induced by authentic peroxynitrite (25u2003μM) and the peroxynitrite generating agent 3‐morpholinosidnonimine (SIN‐1, 250u2003μM). Preincubation of TPEN with equimolar Zn2+ but not Ca2+ or Mg2+ blocked the cytoprotective effect of the chelator. TPEN (10u2003μM) markedly reduced the peroxynitrite‐induced decrease of mitochondrial transmembrane potential, secondary superoxide production and mitochondrial membrane damage, indicating that it acts proximal to mitochondrial alterations. Although TPEN (1–300u2003μM) did not scavenge peroxynitrite, it inhibited PARS activation in a dose‐dependent manner. The cytoprotective effect of TPEN is only partly mediated via PARS inhibition, as the chelator also protected PARS‐deficient thymocytes from peroxynitrite‐induced death. While being cytoprotective against peroxynitrite‐induced necrotic death, TPEN (10u2003μM), similar to other agents that inhibit PARS, enhanced apoptosis (at 5–6u2003h after exposure), as characterized by phosphatydilserine exposure, caspase activation and DNA fragmentation. In conclusion, the current data demonstrate that TPEN, most likely by zinc chelation, exerts protective effects against peroxynitrite‐induced necrosis. Its effects are, in part, mediated by inhibition of PARS.

BCL-2 protects peroxynitrite-treated thymocytes from poly(ADP-ribose) synthase (PARS)-independent apoptotic but not from PARS-mediated necrotic cell death

In thymocytes, peroxynitrite induces poly(ADP-ribose) synthetase (PARS) activation, which results in necrotic cell death. In the absence of PARS, however, peroxynitrite-treated thymocytes die by apoptosis. Because Bcl-2 has been reported to inhibit not only apoptotic but also some forms of necrotic cell death, here we have investigated how Bcl-2 regulates the peroxynitrite-induced apoptotic and necrotic cell death. We have found that Bcl-2 did not provide protection against peroxynitrite-induced necrotic death, as characterized by propidium iodide uptake, mitochondrial membrane potential decrease, secondary superoxide production, and cardiolipin loss. In the presence of a PARS inhibitor, peroxynitrite-treated thymocytes from Bcl-2 transgenic mice showed no caspase activation or DNA fragmentation and displayed smaller mitochondrial membrane potential decrease. These data show that Bcl-2 protects thymocytes from peroxynitrite-induced apoptosis at a step proximal to mitochondrial alterations but fails to prevent PARS-mediated necrotic cell death. Activation of tissue transglutaminase (tTG) occurs in various forms of apoptosis. Peroxynitrite did not induce transglutaminase activity in thymocytes and did not have a direct inhibitory effect on the purified tTG. Basal tTG was not different in Bcl-2 transgenic and wild type cells.

Local and systemic inflammation: role of poly (ADP-ribose) synthetase activation by reactive nitrogen species

Poly (ADP)-ribosyltransferase (PARS), also known as poly(ADP-ribose) polymerase (PARP) is an abundant nuclear enzyme present throughout the phylogenetic spectrum [1]. The precise physiologic roles of PARS remain undefined: its traditional role as a DNA-repair enzyme has been questioned by recent studies [2]. PARS appears to play diverse roles, participating in DNA repair [3, 4], chromatin relaxation [5], cell differentiation [6], DNA replication [7], transcriptional regulation [8], control of cell cycle [9], p53 expression and apoptosis [10], and transformation [11]. In the last decade, the novel concept emerges that under various pathophysio-logical conditions, PARS plays a crucial role in the regulation and generation of the inflammatory response. Here, we summarize the evidence favoring this new role for PARS in various forms of local and systemic inflammation and propose therapeutic opportunities afforded by PARS inhibition.